The present invention relates to a novel protein having a homology with ribonucleotide reductase, to the DNA coding the protein and so forth.
Recent developments of research have proven that cancer is a genetic disease in which multiple genetic mutations accumulate in somatic cells, resulting in uncontrolled cell growth. p53 is the tumor suppressor gene in which abnormalities have been found most frequently in human cancers, and has been clarified to have a variety of physiological functions including induction of G1 arrest and apoptosis, and checkpoint control when DNA is damaged. It is thought that these functions are exercised when p53 acts as a transcription factor to control the expression of the target gene. It is suggested that p53 abnormalities are associated with cancer malignancy, resistance to anti-cancer agents and radiation therapy, metastasis and vascularization (The New England Journal of Medicine Vol. 329 p. 1318 (1993), etc.).
Ribonucleotide reductase is a rate-limiting enzyme, which converts ribonucleotides to their corresponding deoxyribonucleotides and supplies them for purposes of DNA synthesis (Science Vol. 260 p. 1773 (1993)). This enzyme is a heterodimer comprising a large subunit (R1) and a small subunit (R2), with both R1 and R2 (National Center for Biotechnology Information GenBank Accession No. X59618) consisting of homodimers. Enzyme activity is controlled by the amount of R2. The amount of R2 is controlled depending on the cell cycle, with the most being highly expressed during the S period. Ribonucleotide reductase has been well studied in yeasts, and three subunits (RNR1, RNR2, RNR3) are known to exist in yeasts. RNR1 and RNR3 correspond to R1 in mammals and RNR2 to R2 in mammals, and control of RNR1 expression is dependent on cell cycle. It has been reported that when DNA is damaged by irradiation for example, RNR1 expression is not induced, but induction of RNR3 expression increases more than 100 fold (Genes and Development Vol. 4 p. 740 (1990)). It has also been reported that expression of R2 is greater in highly malignant cancer cells, which are resistant to anti-cancer agents and radiation therapy (Biochemistry and Cell Biology Vol. 68 p. 1364 (1990)).
The TP53R2H gene obtained in the examples below has a ribonucleotide reductase signature sequence, which has high homology with R2 and is conserved among species, and the product of the TP53R2H gene may be involved in supplying deoxyribonucleotide for DNA synthesis. Since expression of TP53R2H is also induced by DNA damage due to anti-cancer agent treatment or irradiation (not the case with R2), it may be involved particularly in supplying deoxyribonucleotides during DNA repair following DNA damage. It can therefore be expected that blocking of TP53R2H in cases of highly malignant cancer with resistance to anti-cancer agents and radiation therapy would be a highly effective treatment with few side-effects. Moreover, because TP53R2H may act as a homodimer (as R2 does), it might be possible to confer a dominant negative effect and suppress the activity of the enzyme by introducing mutated TP53R2H genes or TP53R2H protein into cancer cells, so both mutated TP53R2H genes and TP53R2H protein should be useful as therapeutic agents for highly malignant cancers with resistance to anti-cancer agents and radiation therapy. In addition, investigation of mutations to this gene should be useful in cancer diagnosis and prevention.
As a result of dedicated research, the present inventors succeeded in cloning, from a human muscle-derived cDNA library, cDNA which codes for a novel protein having a homology with ribonucleotide reductase, and perfected the present invention as a result of further study based on these findings.
That is, the present invention relates to:
(1) A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof;
(2) The protein according to (1) above, having ribonucleotide reductase activity;
(3) A partial peptide of the protein according to (1) above, or a salt thereof;
(4) DNA comprising DNA having a base sequence encoding the protein according to (1) above or the partial peptide according to (3) above;
(5) DNA according to (4) above, having the base sequence represented by SEQ ID NO: 2;
(6) DNA according to (4) above, having the base sequence represented by SEQ ID NO: 12;
(7) A recombinant vector comprising the DNA according to (4) above;
(8) A transformant transformed by the recombinant vector according to (7) above;
(9) A method for manufacturing the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above, wherein the transformant according to (8) above is cultured to produce the protein or salt thereof according to (1) above or partial peptide or salt thereof according to (3) above, which is then accumulated and collected;
(10) A pharmaceutical comprising the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above;
(11) A pharmaceutical comprising the DNA according to (4) above;
(12) An antibody to the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above;
(13) A diagnostic agent comprising the antibody according to (12) above;
(14) A method for screening a compound or salt thereof which inhibits or activates the enzyme activity of the protein or salt thereof according to (1) above, characterized by the use of the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above;
(15) A kit for screening a compound or salt thereof which inhibits or activates the enzyme activity of the protein or salt thereof according to (1) above, comprising the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above;
(16) A compound or salt thereof which inhibits or activates the enzyme activity of the protein or salt thereof according to (1) above, obtained by using the screening method according to (14) above or the screening kit according to (15) above;
(17) A pharmaceutical comprising the compound or salt thereof according to (16) above, and;
(18) The pharmaceutical or the like according to (17) above, wherein the pharmaceutical is a pharmaceutical for prevention or treatment of cancer.
Moreover, the present invention also provides:
(19) DNA comprising DNA having a base sequence which is hybridizable under highly stringent conditions with the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 12;
(20) A recombinant vector comprising the DNA according to (19) above;
(21) A transformant transformed by the recombinant vector according to (20) above;
(22) A method for manufacturing the protein or salt thereof encoded by the DNA according to (19) above, wherein the transformant according to (21) above is cultured to produce the protein encoded by the DNA according to (19) above, which is then accumulated and collected;
(23) The protein or salt thereof encoded by the DNA according to (19) above, produced by the method according to (22) above;
(24) A method for quantifying the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above in a partial peptide or salt thereof according to (3) above in a test solution, wherein the antibody according to (12) above is reacted competitively with a test solution and the labeled protein according to (1) above or partial peptide according to (3) above or salts of these, and the proportion of the labeled protein or salt thereof according to (1) above or partial peptide or salt thereof according to (3) above, which binds to the antibody is measured.
(25) A method for quantifying the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above in a test solution, wherein the test solution is reacted either simultaneously or consecutively with an antibody according to (12) above which has been insolubilized on a carrier and another, labeled antibody according to (12) above, and the activity of the labeling agent on the insolibilizing carrier is measured;
(26) A pharmaceutical comprising the antibody according to (12) above (preferably, an antibody which acts to neutralize the activity of the protein according to (1) above);
(27) The pharmaceutical according to (26) above which is a therapeutic agent for cancer;
(28) A method for screening a compound or salt thereof which inhibits or activates the enzyme activity (such as ribonucleotide reductase activity or the like) of the protein or salt thereof according to (1) above, wherein the enzyme activity of the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above is measured and compared when (i) a substrate is brought into contact with the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above, and when (ii) a substrate and a test compound are brought into contact with the protein or salt thereof according to (1) above or the partial peptide or salt thereof according to (3) above.
(29) An antisense oligonucleotide having a base sequence, which is complementary or substantially complementary to the DNA according to (1) or (19) above, and having an action that can control the expression of said DNA;
(30) The antisense oligonucleotide according to (29) above, wherein the base sequence which is substantially complementary to the DNA according to (1) or (19) above is a base sequence having about 90% or greater (preferably about 95% or greater) homology with all or part of a base sequence which is complementary to said DNA.
(31) A pharmaceutical comprising the antisense oligonucleotide according to (29) or (30) above; and
(32) A pharmaceutical according to (31) above which is a therapeutic agent for cancer.